of the developing brain (fig. S9A). The localization of the virus to the ventricle allowed for
selective targeting of the viral construct to post-mitotic cells that had not yet, at the time of
infection, migrated out of the ventricular zone
where they were born (fig. S9A). Using a double
viral-BrdU approach, we found that the method
was specific in targeting cells born on the day of the
viral injection in MEC-L2, within a time window
of 24 to 32 hours (fig. S9, B and D). The method was
validated on sections from visual cortex, where,
as expected, neurogenesis showed an inside-out
progression, with E12 injections only labeling
cells in the deepest layers and E16 injections
targeting exclusively cells in superficial layers
(fig. S9E) (30, 31). In MEC, the topographical
distribution and dorsoventral progression of neurogenesis were identical in BrdU and virally identified cohorts (fig. S9F).

Using the viral approach, we were able to testthe hypothesis that activity in stellate but notpyramidal cells is necessary for driving matu-ration throughout the entorhinal-hippocampalcircuit. We first injected a Cre virus (AAV1-CaMKII-Cre) into the lateral ventricle at either E12 or E16to prime isochronic cohorts of cells to express therecombinase Cre. Then, at P1, we targeted theinhibitory DREADD hM4D(Gi) to primed cellsby injecting the Cre-dependent virus in MEC-L2.Finally, during multiple time windows of post-natal maturation, we delivered CNO through os-motic minipumps, as in the previous experiments(Fig. 5A). Both cohorts of animals received CNOduring a time window of 3 days (w1, P14 to P17;w2, P17 to P20; w3, P20 to P23; w4, P23 to P26).We silenced early-born neurons (E12-labeled)during early (w1 and w2) and late (w3) windowsof maturation (fig. S11) and late-born neuronsduring early (w2) and late (w3 and w4) windows(fig. S12). As expected, in the most dorsal part ofthe MEC, the composition of the labeled cohortwas dominated by stellate cells when the injec-tion was performed at E12 (96% of infected cellscolocalized with reelin; Fig. 5B and fig. S10A). Incontrast, pyramidal cells predominated when theviral mix was injected at E16 (92% of infectedcells colocalized with calbindin; Fig. 5B and fig.

2.124, P > 0.30; fig. S12, A and B). Collectively,
these experiments suggest that the activity in
stellate but not pyramidal cells is necessary for
the maturation of the local network in MEC-L2.

The localization of the instructive signal in
MEC to stellate cells raises the question of whether activity from isochronic stellate cells might
also be necessary for the sequential maturation
across subdivisions of the transverse entorhinal-hippocampal circuit. Silencing isochronic stellate
cells was sufficient to prevent the maturation-associated down-regulation of DCX in every area
of the network (all comparisons with Student’s t
test were significant, t > 7.44 and P < 0.01, with
the exception of LEC-L2, t = 0.36 and P = 0.7,
where most cells were in an immature state under
the control condition too; Fig. 5C and fig. S13A).
Silencing stellate cells was also sufficient to decrease PV expression throughout the entorhinal-hippocampal loop (all comparisons were significant,
t > 6.23 and P < 0.01, with the exception of LEC-
L5 and LEC-L2, t < 0.13 and P > 0.15; fig. S13B,
left). Synaptogenesis was similarly retarded (all
comparisons were significant, t > 4.71 and P <
0.01; fig. S13B, right). In striking contrast, silencing isochronic pyramidal cells did not exert any
effect on maturation of the transverse entorhinal-hippocampal circuit (t < 1.03 and P > 0.34; Fig. 5D
and fig. S13, A and C). We also silenced stellate
cells at later time points, between P20 and P23,
after the maturation of their postsynaptic partners is complete. This had no effect on DCX expression at any stage of the entorhinal-hippocampal
network (t < 2.41 and P > 0.72; fig. S13D), suggesting that the instructive role that stellate cells
have on entorhinal-hippocampal circuit maturation is temporally limited. Together, the results
identify stellate cells as the source of the activity-dependent instructive signal that drives the sequential maturation of the entorhinal-hippocampal
network.

Isochronic cohorts of neurons
act synergistically

By exploiting our method to genetically label isochronic cohorts of neurons, we were able to show
Donato et al., Science 355, eaai8178 (2017) 17 March 2017 7 of 10
Fig. 6. Isochronic cohorts of neurons act synergistically to drive MEC-L2 maturation. Quantitative analysis of network maturation based on DCX
expression along the dorsoventral axis of MEC-L2. DCX− pyramidal cells and PV+ interneurons disappear when an isochronic population of E12-born
neurons is silenced (orange), but not when the ligand targets a comparable fraction of randomly labeled L2 cells (green, “Random cells – entire network”:
20% of MEC-L2 excitatory neurons labeled) or stellate cells (blue, “Random cells – stellate cells”: 20% of MEC-L2 stellate cells labeled) (>45,000
neurons from three animals per group). Three categorically different classes of treatment were pooled to form the control group, as in Fig. 2.